The invention""s method for preparing compounds of the general formulas 
wherein R1 is acyl , alkoxycarbonyl or aryloxycarbonyl and R2 is a hydrogen atom or C1-10 alkyl, comprises treating with a hydrolase and an effective amount of a nucleophile and a base in a constant pH range a racemic lactam of the formula 
Formula I compounds, such as, for example, (1R,4S)-2-acetyl-2-azabicyclo[2.2.1]hept-5-ene-3-one are important intermediates for preparing (1R,4S)-1-amino4hydroxymethyl)-2-cyclopentene, which, in turn, is an important intermediate for preparing carbocyclic nucleosides, such as, for example, Carbovir (Campbell et al. J. Org. Chem. 1995, 60, 4602-4616). Formula II compounds, such as, for example, the propyl ester of (1S,4R)-acetylamino-2-cyclopentene-1-carboxylic acid, are an important intermediate for preparing (1S,4R)-1-amino-4-(hydroxymethyl)-2-cyclopentene, which similarly can be an important intermediate for preparing carbocyclic nucleosides.
Only the chemical preparation of (1R,4S)-2-acetyl-2-azabicyclo[2.2.1]hept-5-ene-3-one by acylating (1R,4S)-2-azabicyclo[2.2.1]hept-5-ene-3-one (Katagiri et al., Tetrahedron Letters, 1997, 38, 1961) is known. According to this method, (1R,4S)-2-acetyl-2-azabicyclo[2.2.1]hept-5-ene-3-one can be obtained only from the corresponding (1R,4S)-2-azabicyclo[2.2.1]hept-5-ene-3-one as an educt. This educt is too expensive.
The problem involved in this invention was to develop a method for preparing compounds of general formula I and II, which can be prepared from easily obtainable, inexpensive starting material with good enantiomer purity.
This problem is solved with the new biotechnological method according to claim 1.
The invention""s method for preparing compounds of the general formulas 
wherein R1 is acyl or acyloxy and R2 is a hydrogen atom or C1-10 alkyl, takes place by means of a hydrolase in the presence of a nucleophile and in the presence of a base in a constant pH range starting with a racemic lactam of the formula 
The starting material, the lactam of the general formula III (substrate), can be prepared, for example, according to Taylor et al. (Tet. Asymmetry; 4, 1993,1117).
C1-10 alkyl is linear or branched and substituted or unsubstituted. Examples of C1-10 alkyl are methyl, ethyl, propyl, butyl, isobutyl, t-butyl, isopropyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl and its isomers, as well as chloromethyl, bromomethyl, dichloromethyl, dibromomethyl, chloropropyl and bromobutyl.
Acyl means alkanoyl or arylcarbonyl. Alkanoyl is suitably C1-4 alkanoyl, which can be substituted or unsubstituted. Substituted C1-4 alkanoyl in the following is understood to be substituted with one or more halogen atoms. Examples of C1-4 alkanoyl are acetyl, propionyl, butyryl, chloroacetyl, bromoacetyl and dichloroacetyl. Arylcarbonyl is suitably benzylcarbonyl or phenylcarbonyl, substituted or unsubstituted.
Acyloxy means alkoxycarbonyl or aryloxycarbonyl. Alkoxycarbonyl is suitably C1-4 alkoxycarbonyl, such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl or t-butoxycarbonyl (BOC). Aryloxycarbonyl is suitably benzyloxycarbonyl or phenyloxycarbonyl.
R1 is preferably C1-4 alkanoyl or C1-4 alkoxycarbonyl, in particular acetyl or ethoxycarbonyl.
Hydrolases that can be used are proteases or lipases, preferably proteases, such as serine proteases. Examples of serine proteases that can be used are chymotrypsin, trypsins and subtilisins (bacterial serine proteases). Subtilisins that can be used are commercial subtilisins, such as subtilisin A, subtilisin B, alcalases, ALK enzyme bacillopeptidase A, bacillopeptidase B, bioprases, colistinases, esperases, genenase I, kazusase, maxacal, maxatases, nagarses, peptidases, protease S, protease VIII, protease XXVII, proteinases, such as the alkaline proteinase of Bacillus subtilis or Aspergillus oryzae, proteinase K from Tritirachium album, savinases, subtilopeptidasen, superases, and thermoases. Conducting the biotransformation by means of savinases is preferred. Suitable savinases are savinase 12 Type W(trademark), savinase 16.0 L Type EX(trademark), savinase 32.0 L Type EX(trademark), savinase 4.0 T Type W(trademark), and savinase 8.0 L(trademark). The lipase that can be used is, for example, lipase from Candida Antarctica.
If the hydrolases used are proteases, such as proteases from Bacillus subtilis, proteases from Aspergillus oryzae, proteinase K from Tritirachium album, the (1S, 4R) enantiomer in the racemic lactam of formula III is hydrolyzed suitably into the corresponding compound of general formula II, whereby the (1R, 4S) enantiomer of general formula I is obtained. If the hydrolases used are lipases, such as lipase from Candida Antarctica, the (1R, 4S) enantiomer in the racemic lactam of formula III is hydrolyzed suitably into the corresponding compound of general formula II, whereby the (1S, 4R) enantiomer of general formula I is obtained.
Water or C1-10 alcohols can be used as the nucleophile. Suitable C1-10 alcohols are methanol, ethanol, propanol, isopropanol, butanol, t-butanol, isobutanol, pentanol, hexanol, heptanol, octanol, nonanol or decanol. If the nucleophile used is a C1-10 alcohol, the corresponding ester of general formula II (R2=C1-10 alkyl) is formed, as the expert knows. If water is used as the nucleophile, obviously, the corresponding acid of general formula II (R2=H) is formed.
Depending on the hydrolase and the substrate (formula III lactam), the biotransformation is conducted suitably between pH 5 and 12, preferably between pH 6 and 8. In the invention, for a given hydrolase and a given substrate, the pH value is maintained constant in the presence of a base. The pH value is suitably maintained constant at +/xe2x88x920.5 pH units by addition of a base. If, for example, the substrate is racemic 2-acetyl-2-azabicyclo[2.2.1]hept-5-ene-3-one (R1=acetyl) and savinase is used as the hydrolase, the pH value is held constant at preferably between pH 7.0 and pH 7.5.
An inorganic or organic base can be used as the base. For example, KOH and NaOH are suitable as inorganic bases. A suitable organic base can be, for example, triethanolamine dissolved in an organic solvent. If one of the aforesaid alcohols is used as the nucleophile, the corresponding alcoholate can serve as the base.
The biotransformation is suitably conducted in water, a buffer solution, a C1-10 alcohol or in a mixture of these with an aprotic organic solvent. Suitable aprotic organic solvents are, for example, ether and aromatic hydrocarbons. Tetrahydrofuran, dioxane or t-butyl methyl ether can be used as the ether. Toluene and benzene are suitable aromatic hydrocarbons. The buffer solutions used can be, for example, low molarity, such as 10-100 mM sodium or potassium phosphate buffer, hepes buffer. The C1-10 alcohols used can be those previously described.
The biotransformation can also be conducted so that the lactam of general formula III serves as solvent. Then the biotransformation is suitably conducted in the presence of half of the stoichiometric quantities of water or the corresponding alcohol.
Depending on the solvent, the biotransformation can be conducted in a two-phase or one-phase system. The biotransformation is advisedly conducted in a one-phase system.
After a usual conversion time of a few hours depending on the selected starting material, the desired optically active compounds of general formulas I and II are obtained in outstanding yields and enantiomer purity. The preferred starting materials are racemic 2-acetyl-2-azabicyclo-[2.2.1]hept-5-ene-3-one (R1=acetyl) and the racemic 2-ethoxycarbonyl-2-azabicyclo-[2.2.1]hept-5-ene-3-one (R1=ethoxycarbonyl). The preferred compounds of formula II are (1S, 4R)-4-acetylamino-2-cyclopentene-1-carboxylic acid (R1=acetyl, R2=H), (1S, 4R)-4-ethoxycarbonylamino-2-cyclopentene-1-carboxylic acid (R1=ethoxycarbonyl, R2=H), (1S, 4R)-4-acetylamino-2-cyclopentene-1-carboxylic acid methyl ester (R1=acetyl, R2=CH3), (1S, 4R)-4-acetylamino-2-cyclopentene-1-carboxylic acid butyl ester (R1=acetyl, R2=C4H9), (1S. 4R)-4-acetylamino-2-cyclopentene-1-carboxylic acid ethyl ester (R1=acetyl, R2=C2H5), and (1S, 4R)-4-acetylamino-2-cyclopentene-1-carboxylic acid propyl ester (R1=acetyl, R2=C3Hxe2x80x94). The (1S, 4R)-4-acetylamino-2-cyclopentene-1-carboxylic acid C2-10 alkyl esters, preferably the (1S, 4R)-4-acetylamino-2-cyclopentene-1-carboxylic acid ethyl ester and the (1S, 4R)-4-acetylamino-2-cyclopentene-1-carboxylic acid propyl ester of the formula II are not described in the literature and are similarly part of the invention.
Another part of the invention is the subsequent reaction, the reduction of compounds of general formula I to an optically active 1-amino-4-(hydroxymethyl)-2-cyclopentene derivative, in particular to a (1R,4S)-1-amino-4-(hydroxymethyl)-2-cyclopentene derivative of the general formula 
wherein R1 is as already named.
The reduction is suitably conducted with binary or complex metal hydrides of the boron or aluminum group, such as alkali metal borohydrides, alkaline earth metal borohydrides, alkali metal aluminum hydrides, alkaline earth metal aluminum hydrides.
The binary alkali metal borohydrides or alkaline earth metal borohydrides used can be NaBH4, LiBH4, KBH4, NaAlH4, LiAlH4, KAlH4, Mg(BH4)2, Ca(BH4)2, Mg(AlH4)2, Ca(AlH4)2. Complex metal hydrides of the boron or aluminum group can have the general formula M1M2H4Lm, wherein n is a whole number from 1 to 4, m is a whole number from 4 to 4-n, M1 is an alkali metal atom, M2 is boron or aluminum, and L is C1-4 alkyl, C1-4 alkenyl, C1-4 alkoxy, CN or an amine, or the complex metal hydrides can have the general formula M2HOLp, wherein M2 is as already named, o is a whole number from 0 to 3, and p is a whole number from 3 to 3-o. The M1M2HnLm used can be LiBH(C2H5)3, LiBHx(OCH3)4-n, wherein x is a whole number from 1 to 3. LiAlH(OC(CH3)3)3, NaAlH2(OC2H4OCH3)2, NaAlH2(CH2H5)2 or NaBH3CN. The reduction is conducted preferably with a metal borohydride, such as sodium borohydride.
The metal hydrides are used suitably in a molar ratio of 0.5 to 1per mole of compound of general formula I.
The reduction is conducted suitably under an inert gas atmosphere, such as, for example, under an argon or nitrogen atmosphere.
The reduction can be conducted at a temperature of xe2x88x9210 to 30xc2x0 C., preferably 0 to 10xc2x0 C.
Secondary or tertiary alcohols are suitable as solvents for the reduction. 2-Butanol can be used, for example, as the secondary alcohol, and t-amyl alcohol, for example, as the tertiary alcohol. A secondary alcohol is preferred.
The subsequent conversion, the hydrolysis of the optically active 1-amino-4-(hydroxymethyl)-2-cyclopentene derivatives of formula IV to the corresponding optically active 1-amino4-(hydroxymethyl)-2-cyclopentenes or their salts of the formula 
with an alkaline earth metal hydroxide, alkali metal hydroxide or with a mineral acid is similarly part of the invention. In particular, the (1R,4S)-1-amino-4-(hydroxymethyl)-2-cyclopentene derivative is hydrolyzed to (1R,4S)-1-amino-4-(hydroxymethyl)-2-cyclopentene.
Lithium, sodium or potassium hydroxide is suitable as the alkali metal hydroxide. Barium hydroxide, for example, can be used as the alkaline earth metal hydroxide. The hydrohalide acids such as, for example, hydrochloric acid or hydrobromic acid are suitable as the mineral acids.
The hydrolysis is suitably conducted at a temperature of 50 to 120xc2x0 C., preferably 90 to 100xc2x0 C.
Suitable salts of the (1R,4S)-1-amino-4-(hydroxymethyl)-2-cyclopentene (formula V) are its hydrohalide salts, such as hydrochlorides or hydrobromides.